Molecular analysis of Candida glabrata clinical isolates.

Candida glabrata is an important human pathogen, and an understanding of the genetic relatedness of its clinical isolates is essential for the prevention and control of fungal infections. In this study, we determined the relatedness of 38 Candida glabrata clinical isolates originating from two teaching hospitals in Slovakia. The 14 different genotypes were found by using microsatellite marker analysis (RPM2, MTI and Cg6) and DNA sequencing for analysis of the entire ERG11 gene. Subsequent sequencing of amplified DNA fragments of the PDR1, NMT1, TRP1 and URA3 loci in ten selected clinical isolates revealed identical DNA sequence profiles in five of them. They displayed the same microsatellite marker sizes and contained the same H576Y amino acid substitution recently described in the Pdr1p multidrug resistance transcription factor responsible for azole resistance. These results demonstrate the genetic diversity of C. glabrata clinical isolates in our hospitals and indicate a common clonal origin of some drug resistant ones.

Mutations in the CgPDR1 and CgERG11 genes in azole-resistant Candida glabrata clinical isolates from Slovakia.

Candida glabrata is an important human pathogen that is naturally less susceptible to antimycotics compared with Candida albicans. Ten unmatched C. glabrata clinical isolates were selected from a collection of isolates exhibiting decreased susceptibilities to azole antifungals. Overexpression of the CgPDR1 gene, encoding the main multidrug resistance transcription factor, and its target genes CgCDR1 and CgCDR2, coding for drug efflux transporters, was observed in six fluconazole-resistant isolates. Sequence analysis of the polymerase chain reaction (PCR)-amplified DNA fragments of each isolate's CgPDR1 gene was used to identify two novel L347F and H576Y mutations in CgPdr1p. These proved to be responsible for fluconazole resistance in transformants of the C. glabrata pdr1Delta mutant strain. Five isolates harbouring the H576Y mutation also contained the mutation E502V in CgErg11p 14C-lanosterol-demethylase. Heterologous expression of the CgERG11 mutant allele did not provide evidence for its involvement in azole resistance. In four fluconazole-sensitive isolates that were itraconazole-resistant, slightly enhanced CgCDR2 expression was observed. No upregulation of the CgERG11 gene was observed in any of the ten isolates. The results demonstrate that decreased susceptibilities of C. glabrata clinical isolates to azole antifungals mainly results from gain-of-function mutations in the gene encoding the CgPdr1p transcription factor.